With the advance in developmental engineering technology and the rapid compilation of knowledge in molecular biology, it has become possible to artificially manipulate genes and introduce them to animal individuals [Gordon, J. W. et al., Proceedings of National Academy of Science, U.S.A., Vol. 77, pp. 7380-7384, (1980)]. Thanks to development of various methods of artificially adding exogenous gene essentially not conferred upon the organism of interest, or artificially suppressing expression of endogenous genetic characters essentially conferred upon the organism, a variety of transgenic animals have been generated and reported.
Such transgenic animals are important in clarifying the functions of various cloned genes isolated by gene engineering and other technologies, since they enable research on an individual basis in the functional analysis of genes, regarding which information availability has been limited due to the fact that only cells cultured in vitro, such as cultured cells or primary cultured cells, have been available. Analysis of the physiological functions of cloned genes in vivo, and in particular experiments and studies using such transgenic animals as models of genetic diseases are actively being undertaken.
Embryonic stem cells (ES cells) of a strain established from an inner cell mass of the blastocyst, or the fertilized embryo at the early stage, are capable of being grown and cultured with keeping in an undifferentiated state. These cells possess multipotency, by which they can differentiate into every kind of cell; when injected to a normal early stage embryo, they can participate in embryo formation to form a chimeric animal [Evans M. J. and Kaufman M. H., Nature, Vol. 292, p. 154 (1981)].
Making use of such nature, there have been attempts at creating various gene mutant animals. This trend dates back to 1981, when the ES cell line was established by Evans and Kaufman, followed by extensive research starting at the creation of an ES chimeric mouse by Bradley et al. [Nature, Vol. 309, p. 255 (1984)]. As successful achievements were reported, including homologous recombination in ES cells by Thomas and Capecchi [Cell, Vol. 51, p. 503 (1987)] and subsequent germ line transmission of ES cells by three research groups, including that of Koller et al. [Proceedings of National Academy of Science, U.S.A., Vol. 86, p. 8927 (1989)], there have been rapid advances in the generation of gene-deficient mice and in research using them.
In addition to the EK cells of Evans and Kaufman (ibid.), established ES cell lines reported so far include the ES-D3 cells of Doetschman [Journal of Embryology and Experimental Morphology, Vol. 87, p. 27 (1981)], the CCE cells of Robertson [Nature, Vol. 323, p. 445 (1986)] and the BL/6 III cells of Ledermann and Burki [Experimental Cell Research, Vol. 197, p. 254], most of which have been established from 129 strain mouse embryos. Despite the very high value of use in gene targeting, which modifies a particular gene, and for other purposes, ES cells are now subject to limitation as to applicability because their establishment, and their passage while in an undifferentiated state, are difficult. To make routine the creation of animal disease models using ES cells, it is necessary to develop a system for constantly establishing and supplying good ES cells.
Animals deficient in gene expression generated using such ES cells include the HPRT-gene-deficient mouse generated using a spontaneously mutated ES cell by Hooper et al. [Nature, Vol. 326, p. 292 (1987)] and Knehn et al. [Nature, Vol. 326, p. 295 (1987)], the p53-deficient mouse, which lacks p53, a tumor suppressor gene, generated by Donehower et al. [Nature, Vol. 356, p. 215 (1992)], the .beta.2-microglobulin gene mutant mouse generated by Zijlstra et al. [Nature, Vol. 344, p. 742 (1990)], the RAG-2 (V(D)J recombination activation gene) mutant mouse, an immune disease model mouse, generated by Sinkai et al. [Cell, Vol. 68, p. 855 (1992)], the MHC class II mutant mouse generated by Glimcher et al. [Science, Vol. 253, p. 1417 (1991)] and Cosgrove et al. [Cell, Vol. 66, p. 1051 (1991)], the int-1-deficient mouse, a development/growth-related disease model mouse generated by MacMahon et al. [Cell, Vol. 62, p. 1073 (1990)], and the src-deficient mouse, which develops symptoms like those of marble bone disease, generated by Soriano et al. [Cell, Vol. 64, p. 693 (1991)].
The neurotrophins comprise factors with sequence similarity [e.g., nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), NT-4 and NT-5]. Since the discovery of NGF by Levi-Monntalchini [Annual of New York Academy of Science, 55, 330 (1952)] and Cohen et al. [Proceedings of National Academy of Science, U.S.A., Vol. 40, p. 1014 (1954)], BDNF [Barde Y-A et al., EMBO Journal, Vol. 1, pp. 549-553 (1982)], NT-3 [Hohn et al., Nature, Vol. 344, p. 399 (1990); PCT Patent Publication No. WO91/03569], NT-4 [Neuron, Vol. 6, p. 845 (1991)] and NT-5 [Neuron, Vol. 7, p. 857 (1991)] have been reported one by one, drawing attention from those searching for preventive or therapeutic measures against diseases of the cranial nervous system, particularly dementia.
These factors have been found to play various role such as nerve cell differentiation, maturation, survival, functional retention and proliferation, and are classified under a single family because they are structurally similar to each other. They are known to show distinct patterns of expression during stages of development, and to act on different types of cells while sharing a role in the survival of several kinds of primary culture nerve cells. Concerning their actions, it is known that NGF shows activities on peripheral sympathetic nerve cells and septal cholinergic nerve cells, and that BDNF acts on motor nerve cells and midbrain dopaminergic nerve cells. As for actions on other factors and cells of non-nerve tissue, much remains unknown, though NGF is known to promote peripheral lymphocyte colony formation.
As for NT-3, human nerve growth factor 2 (NGF-2), identical to NT-3, is disclosed as a polypeptide (I) in European Patent Publication No. 386,752, and reported in FEBS Letters, Vol. 266, pp. 187-191 (1990). It is known that (1) it expresses markedly in the kidney, cerebral hippocampus and cerebellum, (2) it expresses more strongly in neonates than in mature animals, and (3) it acts on nerve cells on which NGF and BDNF show little or no activity (e.g., nerve cells of nodose ganglion origin). These facts suggest that NGF-2/NT-3 plays an important role in the development of the nervous system. Also, this factor has recently been shown to promote human peripheral lymphocyte colony formation, suggesting that it also plays an important role in the growth of leukocytes, neutrophils etc. However, much remains to be clarified as to the function of NT-3 in vivo, including these actions. Although an animal model deficient in NT-3 expression (which produces no NT-3, or only a trace amount) is desirable as a model for elucidation of the factions of NT-3 in vivo, no such animal models have been generated.